5 research outputs found

    Influence of ß-cryptogein transgene on catalase and guaiacol-peroxidase activity in Coleus blumei hairy roots

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    Svojstvo biotičkih elicitora da potiču obrambene odgovore u biljkama koristi se u in vitro kulturi kao strategija za povećanu proizvodnju komercijalno važnih spojeva. Elicitori se također koriste i u svrhu poboljšanja otpornosti biljke na patogene i neke okolišne stresove. Beta-kriptogein je proteinski elicitor koji izlučuje fitopatogena oomiceta Phythophthora cryptogea, koji kod biljaka izaziva hipersenzitivni odgovor i sustavno stjecanje otpornosti protiv nekih patogena. Cilj ovog istraživanja je kontroliranom sintezom kriptogeina u biljci oponašati napad patogenom, te na taj način poboljšati antioksidativni status biljke. U tu svrhu je transgeno korijenje ukrasne koprive (Coleus blumei), u kojem se sintetčki gen crypt nalazi pod kontrolom inducibilnog alkoholnog promotora, tretirano etanolom kako bi se inducirala sinteza ß-kriptogeina. Kao pokazatelji oksidativnog stresa mjerene su količine vodikova peroksida (H2O2) i produkata lipidne peroksidacije (TBARS). Antioksidativni staus je određen mjerenjem aktivnosti katalaze i gvajakol-peroksidaze. Inducibilna ekspresiju gena crypt je povećala aktivnosti antioksidativnih enzima (katalaze i gvajakol-peroksidaze) u kosmatom korijenju ukrasne koprive. Razine H2O2 i TBARS-a su pokazale odusutnost oksidativnog stresa. Povećani antioksidativni status mogao bi poboljšati otpornost biljaka protiv patogena i drugih okolišnih stresova.The property of biotic elicitors to induce plant defence response was used as an strategy for enhancing production of commercially important compounds in plant in vitro cultures. Also, elicitors have been used to improve plant resistance to pathogens and to some environmental stresses. Proteinaceus elicitor secreted by phytophatogenic oomycete Phythophthora cryptogea, ß-cryptogein, induces hypersensitive response and systemic acquired resistance in plants against some pathogens. The aim of this thesis was to mimic pathogen attack by using controlled synthesis of cryptogein, and thus improve antioxidant status of the plant. In order toinduce synthesis of the ß-cryptogein Coleus blumei hairy roots harboring synthetic crypt gene were generated under the control of alcohol-inducible promoter after ethanol treatment. As an indicator of the oxidative stress hydrogen peroxide (H2O2) and lipid peroxidation product (TBARS) contents were estimated. Antioxidative status was determined by measuring catalase and guiacol peroxidase activities. Induced expression of crypt gene caused increase of antioxidant enzymes activities (GPOD and CAT) in Coleus blumei hairy roots. Contents ofH2O2 and TBARS confirmed the absence of oxidative stress. Increased antioxidative status could provide enhance resistance against pathogens and other environmental stresses

    Sudden cardiac death due to deficiency of the mitochondrial inorganic pyrophosphatase PPA2

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    We have used whole exome sequencing to identify biallelic missense mutations in the nuclearencoded mitochondrial inorganic pyrophosphatase (PPA2) in ten individuals from four unrelated pedigrees that are associated with mitochondrial disease. These individuals show a range of severity, indicating that PPA2 mutations may cause a spectrum of mitochondrial disease phenotypes. Severe symptoms include seizures, lactic acidosis and cardiac arrhythmia and death within days of birth. In the index family, presentation was milder and manifested as cardiac fibrosis and an exquisite sensitivity to alcohol, leading to sudden arrhythmic cardiac death in the second decade of life. Comparison of normal and mutated PPA2 containing mitochondria from fibroblasts showed the activity of inorganic pyrophosphatase significantly reduced in affected individuals. Recombinant PPA2 enzymes modeling hypomorphic missense mutations had decreased activity that correlated with disease severity. These findings confirm the pathogenicity of PPA2 mutations, and suggest that PPA2 is a new cardiomyopathy-associated protein, which has a greater physiological importance in mitochondrial function than previously recognized

    Pluripotent stem cell-derived bile canaliculi-forming hepatocytes to study genetic liver diseases involving hepatocyte polarity

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    Background & Aims: Hepatocyte polarity is essential for the development of bile canaliculi and for safely transporting bile and waste products from the liver. Functional studies of autologous mutated proteins in the context of the polarized hepatocyte have been challenging because of the lack of appropriate cell models. The aims of this study were to obtain a patient-specific hepatocyte model that recapitulated hepatocyte polarity and to employ this model to study endogenous mutant proteins in liver diseases that involve hepatocyte polarity. Methods: Urine cell-derived pluripotent stem cells, taken from a patient with a homozygous mutation in ATP7B and a patient with a heterozygous mutation, were differentiated towards hepatocyte-like cells (hiHeps). HiHeps were also derived from a patient with MEDNIK syndrome. Results: Polarized hiHeps that formed in vivo-like bile canaliculi could be generated from embryonic and patient urine cell-derived pluripotent stem cells. HiHeps recapitulated polarized protein trafficking processes, exemplified by the Cu2+-induced redistribution of the copper transporter protein ATP7B to the bile canalicular domain. We demonstrated that, in contrast to the current dogma, the most frequent yet enigmatic Wilson disease-causing ATP7B-H1069Q mutation per se did not preclude trafficking of ATP7B to the trans-Golgi Network. Instead, it prevented its Cu2+-induced polarized redistribution to the bile canalicular domain, which could not be reversed by pharmacological folding chaperones. Finally, we demonstrate that hiHeps from a patient with MEDNIK syndrome, suffering from liver copper overload of unclear etiology, showed no defect in the Cu2+-induced redistribution of ATP7B to the bile canaliculi. Conclusions: Functional cell polarity can be achieved in patient pluripotent stem cell-derived hiHeps, enabling, for the first time, the study of the endogenous mutant proteins, patient-specific pathogenesis and drug responses for diseases where hepatocyte polarity is a key factor. Lay summary: This study demonstrates that cells that are isolated from urine can be reprogrammed in a dish towards hepatocytes that display architectural characteristics similar to those seen in the intact liver. The application of this methodology to cells from patients diagnosed with inherited copper metabolism-related liver diseases (that is, Wilson disease and MEDNIK syndrome) revealed unexpected and novel insights into patient mutation-specific disease mechanisms and drug responses. (C) 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved

    Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency

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    Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis
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